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ORIGINAL RESEARCH |
From the Division of Perinatology and Gynecology and the Division of Medical Genetics of the University Medical Center Utrecht, Utrecht; and Department of Experimental Immunohematology, Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Address reprint requests to: R. J. P. Rijnders, MD, UMC Utrecht KE 04.123.1 orally. Box 85090 3508 AB Utrecht, The Netherlands; e-mail: r_rijnders{at}hotmail.com.
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ABSTRACT |
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METHODS: Real-time quantitative polymerase chain reactions (PCRs) of rhesus-D sequences and the SRY gene were validated and offered to patients with an enhanced risk for sex-linked fetal pathology and patients with rhesus-D antibodies.
RESULTS: In the validation group, 72 samples were analyzed. Sensitivity of the rhesus-D real-time quantitative PCR in maternal plasma was 100% (95% confidence interval [CI]91.8%, 100%) and specificity was 96.6% (95% CI 82.2%, 99.9%). Sensitivity of the SRY real-time quantitative PCR was 97.2% (95% CI 85.5%, 99.9%), and specificity was 100% (95% CI 88.1%, 100%). The technique was used successfully in a clinical setting in 24 women. Overall, invasive tests were avoided in 41.7% of these patients.
CONCLUSION: Detection of cell-free fetal DNA from maternal plasma is a reliable technique that can substantially reduce invasive prenatal tests.
LEVEL OF EVIDENCE: II-2
The original aim of our study was to validate the real-time quantitative PCR of rhesus-D in plasma from pregnant women taken before amniocentesis or chorionic villus sampling. A SRY real-time quantitative PCR was used to prove amplification of fetal DNA in case of a male fetus. The very promising results from this validation group for both the rhesus-D real-time quantitative PCR and the SRY real-time quantitative PCR prompted us to offer the test to patients who had a medical reason for fetal sex or rhesus-D status determination.
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MATERIALS AND METHODS |
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ABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The blood samples were anticoagulated with edetic acid and were centrifuged at 1,200g for 10 minutes within 24 hours after sampling. Plasma was centrifuged again at 2,400g for 20 minutes, and the supernatant was stored at -30°C until further processing. DNA was isolated from 2 mL of plasma by using the Qiagen minikit (Chatswort, CA) with minor adaptations to the blood and body fluid protocol recommended by the manufacturer. The DNA was eluted from the column with 60 ?L of water. For all patients, the real-time quantitative PCR was performed within 5 hours after DNA isolation. For the first set of 21 cases included in the validation protocol, DNA was isolated only once from 2 mL of plasma, and the rhesus-D and SRY real-time quantitative PCRs were performed in triplicate. However, because the SRY real-time quantitative PCR and to a lesser extent the rhesus-D real-time quantitative PCR showed failures of amplification for 1 of the 3 replicates, we decided to change the protocol. For the subsequent 51 cases, we performed a duplicate DNA isolation. In addition, as a control on the DNA isolation and to exclude the presence of possible PCR inhibitors, a real-time quantitative PCR amplifying an in-house reference gene (albumin) was added and performed in duplicate.6 Because the total volume of eluate limited the number of tests that could be performed and because the original aim of the study was the validation of the RHD real-time quantitative PCR, only the RHD real-time quantitative PCR was performed in triplicate, and the SRY real-time quantitative PCR and albumin real-time quantitative PCR were performed in duplicate. For patients with rhesus-D antibodies, the RHD real-time quantitative PCR was also performed in triplicate, and the SRY real-time quantitative PCR and the albumin real-time quantitative PCR were performed in duplicate. Standard anticontamination procedures were followed.
All real-time quantitative PCRs were performed and analyzed with the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). For fetal rhesus-D–typing, a real-time quantitative PCR–specific for the rhesus-D exon 7 was performed with rhesus-D–specific primers rhesus-D–940S: 5'-GGG TGT TGT AAC CGA GTG CTG-3and rhesus-D–1064AS: 5'-CCG GCT CCG ACG GTA TC-3', and the rhesus-D–specific probe rhesus-D–968: 5'-FAM-CCC ACA GCT CCA TCA TGG GCT ACA A-TAMRA-3'. For sex assignment, a real-time quantitative PCR specific for SRY was performed as previously described with slight modifications.6 Both PCRs reached the maximal theoretical sensitivity, and a positive result was obtained from a single genome equivalent (6.6 pg of DNA). The albumin real-time quantitative PCR was performed as described above.7
Reaction mixtures of 50 ?L contained 25 ?L of the Taqman buffer A with the ROX dye as passive reference (Applied Biosystems). For the rhesus-D real-time quantitative PCR, 300 nM of each primer and 100 nM of probe were used. For the SRY real-time quantitative PCR, 900 nM of primers and 150 nM of probe were used. The rhesus-D real-time quantitative PCR and the SRY real-time quantitative PCR were performed with 9 ?L of isolated DNA, whereas the albumin real-time quantitative PCR was always performed with 3 ?L of isolated DNA. The reaction conditions were 2 minutes at 50°C, 10 minutes at 95°C, followed by 50 cycles of 15 seconds at 96°C and 1 minute at 60°C.
In the validation group, we interpreted the test results as follows. When in at least 2 of 3 replicates performed with the rhesus-D real-time quantitative PCR fetal DNA was amplified, the test result was considered positive. The test result of the rhesus-D real-time quantitative PCR was scored negative if in none of the replicates amplification occurred or if only 1 of the replicates for 1 of the DNA isolations showed a positive result. If both DNA isolations showed amplification in 1 of 3 replicates, the overall typing result was considered inconclusive. An SRY real-time quantitative PCR test result was positive if the 2 replicates were positive. An SRY real-time quantitative PCR test result also was considered positive if only 1 of the 2 replicates of 1 DNA isolation was negative, whereas the 2 replicates of the other DNA isolation were both positive. Both for the rhesus-D and SRY real-time quantitative PCR, we concluded that the total test result was inconclusive if the test results of the duplicate DNA isolations were incongruent.
Predicted rhesus-D status and fetal sex in plasma were compared with rhesus-D status and sex in amniotic fluid or CVS and/or to the sex after birth and rhesus-D serology in umbilical cord blood. Rhesus-D genotyping with fetal DNA isolated from amniotic fluid or chorionic villi was performed by multiplex PCR as described previously.8 Sensitivity, specificity, negative and positive predictive values of fetal sex, and/or rhesus-D status determination were calculated. Exact 95% confidence intervals (CIs) of these values were ascertained with the computer program Confidence Interval Analysis (CIA; British Medical Journal, London, UK).9
In the patient studies, the protocol for fetal sex assignment was as follows. Two DNA isolations were performed from 2 mL of plasma (in total, DNA was isolated from 4 mL of plasma). The SRY real-time quantitative PCR was performed in triplicate for each DNA isolation, and the albumin real-time quantitative PCR was performed in duplicate. Scoring for SRY positivity or negativity was performed as described above. Fetal sex was confirmed with first- or early second–trimester ultrasonography.
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RESULTS |
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illustrates that all rhesus-D–positive children (n = 43) were correctly typed with fetal plasma DNA. In 28 of the 29 rhesus-D–negative children, the plasma real-time quantitative PCR on rhesus-D sequences was negative (Table 1). In one case, a child was typed serologically rhesus-D– negative at birth, whereas the plasma rhesus-D real-time quantitative PCR had predicted rhesus-D positivity. The rhesus-D multiplex real-time quantitative PCR performed with DNA from amniotic fluid of this case showed an aberrant pattern: there was an absence of signal for exon 5. This points to the presence of a variant rhesus-D gene, most likely a rhesus-D 
gene.10,11 The sensitivity of the rhesus-D real-time quantitative PCR in maternal plasma was 100% (95% CI 91.8%, 100%) and specificity was 96.6% (95% CI 82.2%, 99.9%). The positive predictive value of the rhesus-D real-time quantitative PCR was 97.7% (95% CI 88.0%, 99.9%) and negative predictive value 100% (95% CI 87.7%, 100%).
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). The SRY real-time quantitative PCR test result was not positive for any of the 29 female fetuses. In 35 of 36 male fetuses, SRY was amplified in both DNA isolations. In one case of a male fetus, only 1 SRY amplification was positive, leading to a false SRY-negative test result (Table 2). The sensitivity of fetal sexing from maternal plasma was 97.2% (95% CI 85.5%, 99.9%), and the specificity was 100% (95% CI 88.1%, 100%). The positive predictive value of fetal SRY in maternal plasma was 100% (95% CI 90.0%, 100%), and the negative predictive value was 96.7% (95% CI 82.8%, 99.9%).
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). Fifteen of these patients were carriers of X-linked diseases; 4 patients were pregnant with a fetus at risk for congenital adrenal hyperplasia; 3 patients were rhesus-D negative, and had rhesus-D antibodies. They all had a probably heterozygous partner, and in 2 patients, fetal sex or rhesus-D status was determined for other reasons. All patients were informed about the investigational character of the test and, in the case of fetal sexing, the need to confirm fetal sex by ultrasound in early second trimester. The latter is absolutely necessary for confirming fetal sex because the sensitivity of the SRY real-time quantitative PCR in the validation series was not 100%. Furthermore, in carriers of X-linked diseases, the test was offered only in situations in which the diagnosis could be made as well in chorionic villi as in amniotic fluid.
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In pregnancies at risk of delivering a fetus with congenital adrenal hyperplasia, it is important to start maternal dexamethasone treatment as early as possible to prevent virilization of a possibly affected female fetus. Male fetuses, affected or not, do not need this treatment. By early fetal sexing, long, unnecessary treatment can be prevented in half of the cases.3 In addition, invasive prenatal diagnosis can be avoided in case of a male fetus. In 3 of 4 women in our study, CVS procedures were avoided because of SRY real-time quantitative PCR in plasma was positive. For the same reason, 2 of these women stopped dexamethasone medication in the eighth week of gestation. The third woman did not take her medication from the start because she was reluctant to do so. One other gravida who was reluctant to take dexamethasone was convinced to start it after 2 early-gestation plasma samples were negative for the SRY gene, suggesting a female fetus.
In 3 rhesus-D–negative women with rhesus-D antibodies and heterozygous partners, real-time quantitative PCRs for rhesus-D and SRY were performed in plasma. In 2 of these women, rhesus-D and SRY sequences were detected. Their pregnancies were closely monitored with frequent ultrasound and Doppler. In 1 patient, SRY real-time quantitative PCR was positive and rhesus-D real-time quantitative PCR negative at a gestational age of 24 weeks. No extra monitoring of the pregnancy was necessary, and an eventual amniocentesis was avoided. In all, 24 patients’ fetal sex or rhesus-D status was correctly predicted. Overall invasive diagnostic tests were avoided in 41.7% of these patients (10 of 24 patients).
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DISCUSSION |
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),2,3,13–18 fetal rhesus-D status in rhesus-D–negative women,19 and single-gene disorders of paternal origin (Saito et al. Lancet.).20,21
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). The rhesus-D pseudogene is rare among the white population but has a frequency of approximately 7% in the black population. The rhesus-D pseudogene shows a partial deletion resulting in an rhesus-D–negative phenotype. Additional rhesus-D typing assays have been developed that are negative if only the rhesus-D pseudogene is present.22 These real-time quantitative PCRs will increase the positive predictive value of an rhesus-D–real-time quantitative PCR to 100%. The only false-negative result in our study occurred in the validation group. The plasma SRY real-time quantitative PCR of a sample obtained at 16–5/7 weeks showed no SRY amplification in both replicates in the first DNA isolation. In the second DNA isolation, the 2 replicates showed 1 positive and 1 negative signal. For a diagnostic setting, the SRY real-time quantitative PCR is performed with 2 DNA isolations, and amplification is performed in triplicate, minimizing the risk of false-negative tests.
The results in the validation group prompted us to use fetal DNA detection by real-time quantitative PCR for clinical purposes. In case of X-linked recessive disease and in fetuses at risk for congenital adrenal hyperplasia, the number of invasive prenatal tests can be dramatically reduced. In 41.7% of our patients, the technique made invasive prenatal diagnosis superfluous. Furthermore, in one carrier of X-linked myotubular myopathy, fewer chorionic villi were required at CVS.
A question to address is at what gestational age the plasma PCR for fetal sexing is reliable. This has been subject of another study. Sensitivity increases substantially between 5 and 10 weeks and reaches its maximum at 10 weeks of gestational age (Rijnders RJ, van der Luijt RB, Peters ED, Goeree JK, van der Schoot CE, Ploos van Anstel JK, Christiaens GC. Earliest gestational age for fetal sexing in cell-free maternal plasma. Prenat Diagn. In press.) Still, in our series as well as in these described in literature (Table 4
.), false-negative results occur. False-positive results of fetal sex determination in plasma were not described by our group or by others. These findings prompted us to created suggested guidelines for the clinical use of fetal sexing in maternal plasma in fetuses at risk for recessive X-linked diseases or CAH.
In carriers of X-linked recessive disease for whom DNA analysis of the gene-defect is possible in both chorionic villi and amniotic fluid, a SRY real-time quantitative PCR should be performed in 2 maternal plasma samples obtained at gestational ages of 9 and 10 weeks (Figure 1). If both samples test negative, the SRY real-time quantitative PCR should be repeated at 14 and 15 weeks. At the same gestational age, results of the plasma real-time quantitative PCR must be confirmed by ultrasound. This "safety net" is still needed because at this time the negative predictive value of the SRY real-time quantitative PCR is not 100%. When one or more samples are tested positive for the SRY gene or when ultrasound reveals a male fetus, amniocentesis can be performed for DNA analysis.
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). Serial testing is performed to 11 weeks or until male DNA is detected in 2 separate samples. In male fetuses, dexamethasone can be discontinued, and invasive diagnostic tests are unnecessary. As long as no male DNA is detected, dexamethasone treatment is continued, unless analysis of fetal chorionic villi shows that the fetus is an unaffected female. This protocol will result in a 50% reduction of invasive prenatal diagnosis and a substantial reduction of dexamethasone exposition of the fetus. The use of ultrasound for determining a male fetus in the case of congenital adrenal hyperplasia is not reliable, because a phenotypic male fetus can be a virilized female. However, in case of a negative plasma SRY real-time quantitative PCR, it can be used as an ascertaining test of the plasma result.
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Footnotes |
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Received July 15, 2003. Received in revised form September 9, 2003. Accepted September 18, 2003.
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REFERENCES |
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